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Abstract:

Cell cultures of Vaccinium myrtillus configured to grow in suspension
culture in a liquid medium. The cells are derived from one or more V.
myrtillus plant parts, such as an edible plant part (e.g., a leaf part or
a berry part) or a stem part. The cells are adapted to grow to a high
density is a relatively short period of time (e.g., about 7 days). In
addition, the cells are adapted to produce high concentrations of
polyphenols and/or procyanidins and essentially no anthocyanin. Methods
for production of polyphenols and/or procyanidins from Vaccinium
myrtillus cells grown in suspension culture are disclosed.

Claims:

1. A cell culture, comprising: a plurality of friable Vaccinium myrtillus
cells in a suspension cell culture, the cells being derived from one or
more of: a hypocotyl, a cotyledon, a leaf section, a stem section, or a
root section of a seedling; or a berry, a stem section including a node
or an internode, or a leaf section of a mature plant, wherein the
plurality of Vaccinium myrtillus cells are selected to be capable of
obtaining a packed cell volume of at least 55% in 7 days of growth, and
wherein at least 5% of a dry mass of the plurality of Vaccinium myrtillus
cells is comprised of procyanidins.

2. The cell culture of claim 1, wherein the berry includes a berry skin.

3. The cell culture of claim 1, wherein at least 10%, 15%, or 20% of a
dry mass of the plurality of Vaccinium myrtillus cells is comprised of
polyphenols.

4. The cell culture of claim 1, wherein at least 7.5% of the dry mass of
the plurality of Vaccinium myrtillus cells is comprised of procyanidins.

5. The cell culture of claim 1, wherein at least 10% of the dry mass of
the plurality of Vaccinium myrtillus cells is comprised of procyanidins.

6. The cell culture of claim 1, wherein at least 15% of the dry mass of
the plurality of Vaccinium myrtillus cells is comprised of procyanidins.

7. The cell culture of claim 1, wherein the dry mass of the plurality of
Vaccinium myrtillus cells comprises less than 0.5% anthocyanin.

8. The cell culture of claim 1, wherein the dry mass of the plurality of
Vaccinium myrtillus cells comprises less than 0.1% anthocyanin.

9. The cell culture of claim 1, wherein the dry mass of the plurality of
Vaccinium myrtillus cells comprises less than 0.01% anthocyanin.

10. The cell culture of claim 1, wherein the dry mass of the plurality of
Vaccinium myrtillus cells comprises less than 0.001% anthocyanin.

11. The cell culture of claim 1, wherein the cells in the suspension cell
culture are derived from a cell callus of Vaccinium myrtillus.

12. The cell culture of claim 1, wherein the suspension cell culture
includes a granular suspension of cells.

13. The cell culture of claim 1, wherein the suspension cell culture
includes a fine suspension of cells.

14. The cell culture of claim 1, wherein the plurality of friable
Vaccinium myrtillus cells in the suspension cell culture are derived from
an edible plant part.

15. The cell culture of claim 14, wherein the edible plant part is one or
more of a leaf part or a berry part.

16. A cell culture, comprising: a plurality of friable Vaccinium
myrtillus cells in a suspension cell culture, the cells being derived
from an edible plant part, wherein the plurality of Vaccinium myrtillus
cells are selected to be capable of obtaining a packed cell volume of at
least 55% in 7 days of growth, and wherein at least 10%, 15%, or 20% of a
dry mass of the plurality of Vaccinium myrtillus cells is comprised of
polyphenols and at least 5% of a dry mass of the plurality of Vaccinium
myrtillus cells is comprised of procyanidins.

17. The cell culture of claim 16, wherein the edible plant part is one or
more of a leaf or a berry.

18. The cell culture of claim 16, wherein the berry includes a berry
skin.

19. A cell culture, comprising: a plurality of friable Vaccinium
myrtillus cells in a suspension cell culture, the cells being derived
from at least a portion of a berry, wherein the plurality of Vaccinium
myrtillus cells are selected to be capable of obtaining a packed cell
volume of at least 55% in 7 days of growth, and wherein at least 10%,
15%, or 20% of a dry mass of the plurality of Vaccinium myrtillus cells
is comprised of polyphenols and at least 5% of a dry mass of the
plurality of Vaccinium myrtillus cells is comprised of procyanidins.

20. The cell culture of claim 19, wherein the berry portion includes a
berry skin.

21-75. (canceled)

Description:

BACKGROUND

[0001] The extract from the fruit Vaccinium myrtillus (more generally
referred to as bilberry) has long been used for therapeutic purposes. In
Europe it has been used for hundreds of years to treat diarrhea and
dysentery, as well as diseases of the lungs, liver, and stomach. In
addition, it is believed that British fighter pilots in World War II ate
bilberry jam to help improve their night vision. More recently, extracts
from the fruit of the V. myrtillus plant has been shown to possess
potential anti-carcinogenic activity. The clinical benefits of V.
myrtillus as both a dietary supplement and a therapeutic have been
attributed to the presence of abundant amounts of flavonoids and
anthocyanins in Bilberry. These antioxidant compounds scavenge damaging
particles known as free radicals in the body, helping to prevent or
reverse damage to cells. Antioxidants have been shown to help prevent a
number of long-term illnesses such as heart disease, cancer, and macular
degeneration. The V. myrtillus fruit also contains tannins, which are
known to act as both an anti-inflammatory and an astringent.

[0003] Berries of the Vaccinium species have been shown to possess radical
scavenging capacity in various in vitro models using assays of the oxygen
radical absorbance capacities (ORAC), the ferric reducing antioxidant
power (FRAP), the total oxidant scavenging capacity (TOSC), and the free
radical scavenging activity against 2,2-diphenyl-1 -picrylhydrazyl (DPPH)
radical as well as antioxidant capacities in inhibiting oxidation of
methyl linoleate, liposomes, and human low-density lipoprotein (LDL)
(Maata-Riihinen et al). Cultivated cranberry (V. macrocarpon Ait.) and
wild lingonberry contain both A- and B-type procyanidins (Gu et.al.,
Morimoto et.al., Foo et.al.) whereas primary B-type procyanidins were
identified in wild (V. angustifolium Ait.) and cultivated blueberries (V.
corymbosum L., V. ashei L.) (Foo et.al.; Prior et. al.; Schmidt et.al.)
Rare A-type low molecular weight procyanidins were detected in wild
lingonberry, cranberry, bilberry, and bog whortleberry and were present
at higher levels than the more common B-type procyanidins (Maata-Riihinen
et.al.). The rare A-Type procyanidin is known to act as deterrent to
adhesion of bacterial cilia to the endothelial layers helping in
prevention of urinary tract infection (Nowack and Schmidt; Foo et
al2;). It is a general anti inflammatory agent which is known in
Bilberry for many decades to cure intestinal inflammations. In addition,
V. myrtillus leaves have 35 different flavon-3-ols, procyanidins,
flavonols and their glycosides, and various phenolic acid conjugates
(Hokannen et.al.).

[0004] Vaccinium myrtillus is difficult to grow and is therefore rarely
cultivated. As a result, the fruit is generally collected from wild
plants during its limited growing season (May through September), which
must be both wet and warm. Thus, the supply of the berries is unreliable
and the berries are available in limited quantities. Moreover, the fruit
are softer and juicier than the related blueberry, such that they must be
harvested by hand, and are difficult to transport, which contribute to
the high cost of the fresh fruit harvested from the V. myrtillus plant.
Also due to the high demand of the ripe fruit, unripe fruits and leaves
are not economically viable products to collect. These are the parts of
the plant that have highest amounts of the procyanidin. In view of the
clinical benefits of V. myrtillus and the difficulty in cultivating these
plants, there is a need to develop a sustainable in vitro culture system
for the cells of these plants.

SUMMARY

[0005] The present disclosure relates to cell culture of Vaccinium
myrtillus that are configured to grow in suspension culture in a liquid
medium. The cells are derived from one or more V. myrtillus plant parts,
such as an edible plant part (e.g., a leaf part or a berry part) or a
stem part. The cells are adapted to grow to a high density in a
relatively short period of time (e.g., about 7 days). In addition, the
cells are adapted to produce high concentrations of polyphenols and/or
procyanidins and essentially no anthocyanin. Methods for production of
polyphenols and procyanidins from Vaccinium myrtillus cells grown in
suspension culture are also disclosed.

[0006] In one embodiment, a cell culture is described. The cell culture
includes a plurality of friable Vaccinium myrtillus cells in a suspension
cell culture. The cells in suspension culture are derived from one or
more of: a hypocotyl, a cotyledon, a leaf section, a stem section, or a
root section of a seedling; or a berry, a stem section including a node
or an internode, or a leaf section of a mature plant. In one embodiment,
the cells can be derived from an edible plant part, such as a leaf part
or a berry part. The cells are selected to be capable of obtaining a
packed cell volume of at least 55% in 7 days of growth, wherein at least
10% of a dry mass of the plurality of Vaccinium myrtillus cells is
comprised of polyphenols and at least 5% of a dry mass of the plurality
of Vaccinium myrtillus cells is comprised of procyanidins.

[0007] Preferably, at least 12.5%, 15%, 20%, or more of the dry mass of
the plurality of Vaccinium myrtillus cells is comprised of polyphenols.
Preferably, at least 7.5%, 10%, 15%, 20%, or more of the dry mass of the
plurality of Vaccinium myrtillus cells is comprised of procyanidins. It
is also preferred that the mass of cells is essentially free of
anthocyanins. For example, it is preferred that the dry mass of the
plurality of Vaccinium myrtillus cells includes less than 0.5%, 0.1%,
0.01%, 0.001%, or less anthocyanin.

[0008] In another embodiment, a method of producing a cell culture of
Vaccinium myrtillus cells is described. The method includes (1) producing
a cell callus of Vaccinium myrtillus cells derived from one or more of: a
hypocotyl, a cotyledon, a leaf section, a stem section, or a root section
of a seedling; or a berry, a stem section including a node or an
internode, or a leaf section of a mature plant, (2) introducing one or
more cells derived from the callus into a liquid medium, (3) agitating
the one or more cells in the liquid medium, (4) replacing the liquid
medium with a fresh liquid medium or transferring the cells to fresh a
fresh liquid medium to establish a suspension cell culture of Vaccinium
myrtillus, (5) growing the suspension cell culture of Vaccinium myrtillus
to a packed cell volume of at least 55%, and (6) selecting suspension
cell cultures having at least 10% of a dry mass of the plurality of
Vaccinium myrtillus cells comprised of polyphenols and/or at least 5% of
a dry mass of the cells comprised of procyanidins.

[0009] In yet another embodiment, a method of increasing growth of
Vaccinium myrtillus cells in suspension cell culture is described. The
method includes (1) providing a suspension cell culture of Vaccinium
myrtillus cells, (2) culturing the cells in a liquid medium in suspension
culture, and (3) selecting suspension cell cultures having greater than
45% packed cell volume (PCV).

[0010] In one embodiment, the method of increasing growth of Vaccinium
myrtillus cells in suspension cell culture further includes selecting
suspension cell cultures having increased polyphenol and procyanidin
accumulation in response to increased sugar concentration in the liquid
medium. In one embodiment, the sugar concentration in the liquid medium
includes approximately 30-60 g/L sucrose. In one embodiment, procyanidin
accumulation in the cells in suspension culture is increased from about
1-2 g/L of PCV at 20 g/L sucrose to about 3-7 g/L of PCV at 30 g/L
sucrose. In one embodiment, polyphenol accumulation in the cells in
suspension culture is increased from about 2-4 g/L of PCV at 20g/L
sucrose to about 5-10 g/L of PCV at 60 g/L sucrose.

[0011] In still yet another embodiment, a method of increasing polyphenol
production from Vaccinium myrtillus cells in culture is described. The
method includes (1) selecting a plurality of Vaccinium myrtillus cells
adapted to grow in suspension culture, (2) and culturing the cells in
suspension culture in the presence of a sufficient amount of sugar to
increase polyphenol production.

[0012] In one embodiment, the sufficient amount of sugar is the liquid
medium having greater than 20 g/L sugar, 20 g/L to 30 g/L sugar, or
greater than 30 g/L sugar. In one embodiment, the sugar is sucrose. In
another embodiment, the sugar is glucose. In one embodiment, the sugar is
present in an amount sufficient for polyphenol production to increase
above 3 g/L packed cell volume (PCV). In another embodiment, the sugar is
present in an amount sufficient for polyphenol production to increase to
at least 7 g/L packed cell volume (PCV).

[0013] In still yet another embodiment, a method of extracting polyphenols
from Vaccinium myrtillus cells in culture is described. The method
includes (1) selecting a plurality of Vaccinium myrtillus cells adapted
to grow in suspension culture, and (2) extracting polyphenols from the
cells using a solvent, wherein at least 10% of a dry mass of the
plurality of Vaccinium myrtillus cells is comprised of polyphenols and at
least 5% of a dry mass of the plurality of Vaccinium myrtillus cells is
comprised of procyanidins.

[0014] In one embodiment, the solvent includes acetone, acetic acid, and
water. In one embodiment, the solvent includes 70% acetone (v/v) and 0.5%
acetic acid (v/v).

[0015] These and other objects and features of the present disclosure will
become more fully apparent from the following description and appended
claims, or may be learned by the practice of the claims as set forth
hereinafter.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] To further clarify the above and other advantages and features of
the present disclosure, a more particular description of the subject
matter of the disclosure will be rendered by reference to specific
embodiments thereof which are illustrated in the appended drawings. It is
appreciated that these drawings depict only illustrated embodiments of
the disclosure and are therefore not to be considered limiting of its
scope. The subject matter of the disclosure will be described and
explained with additional specificity and detail through the use of the
accompanying drawings in which:

[0017] FIG. 1 shows the consumption of sugar with increasing biomass from
25% initial biomass to 50% in one week.

[0018] FIG. 2 shows the growth (a) RI (b) and production (c) at different
shaker speeds. The 500 ml flasks were all inoculated at 20% PCV and PCV,
RI and production yield was measured after 6 days of growth.

[0020]FIG. 4 shows the HPLC chromatogram of extracts from suspension
cells of Bilberry and cocoa in fluorescence detector mode. The labels 1
through 12 indicate the degree of polymerization of procyanidins,
respectively: 1, monomers; 2, dimers; 3, trimers; 4, tetramers; 5,
pentamers; 6, hexamers; 7, heptamers; 8, octamers; 9, nonamers. This
figure confirms that the peaks in Bilberry are procyanidin oligomers by
the fact that the 2 cell lines were extracted in the same way and are run
under same conditions and they have same retention time for each
oligomer.

[0022] The present disclosure relates to cell culture of Vaccinium
myrtillus that are configured to grow in suspension culture in a liquid
medium. The cells are derived from one or more V. myrtillus plant parts,
such as an edible plant part (e.g., a leaf part or a berry part) or a
stem part. The cells are adapted to grow to a high density is a
relatively short period of time (e.g., about 7 days). In addition, the
cells are adapted to produce high concentrations of polyphenols and/or
procyanidins and essentially no anthocyanin. The subject matter of the
disclosure will be described and explained with additional specificity
and detail through the use of the following Examples.

EXAMPLES

Example 1

Surface Sterilization and Seed Germination

[0023] Vaccinium myrtillus seeds were obtained from Horizon Herbs, Oregon.
Leaves, stem sections and immature berries of V. myrtillus (Erin's
Bilberry) used in this Example and the Examples below were collected from
National Clonal Germplasm Repository (NCGR) in Corvallis, Ore.

[0024] Leaves, stem sections, and immature berries were rinsed in running
water for 20 minutes and rinsed in 75% ethanol for 1 minute. Stems were
then cut into smaller pieces. Then the stems, leaves and immature berries
were washed in 25% sodium hypochlorite (v/v) for 15 minutes followed by 5
rinses in sterile distilled water.

[0025] Seeds (Horizon Herbs, Oregon) were surface sterilized by rinsing
first, in 75% ethanol for 1 minute. Then they were washed in 25% sodium
hypochlorite (v/v) for 15 minutes followed by 5 rinses in sterile
distilled water. Seeds were then suspended in 0.1% agarose and plated
onto 100×25 mm Petri plates (approximately 100 seeds per plate).
They were germinated on MS (Murashige and Skoog) medium (4.43 g/L) with
7g/L agar under a 16 hour light and 8 hour dark photoperiod at 23°
C. for 4 weeks.

Example 2

Callus Induction from Vaccinium myrtillus Seedlings Grown In Vitro

[0026] More proliferative growth and friable callus are very important
characteristics of a successful cell line. This example describes methods
and media conditions which were optimized to initiate and maintain callus
from various explants derived from in vitro grown V. myrtillus seedlings.

[0028] Explants were put on various callus induction media (Table 1).
Plates were kept in darkness at 25° C. First signs of callus
formation were seen after 2 weeks of putting explants on plates with
media VM1445, VM1196, VM1204, and VM1233 (VM1233 is described in Madhavi
et al., Plant Science, 131:95-103, 1998). Callus induction rates were
83%, 85%, 85% and 70% respectively. However, callus produced on media
VM1196 and VM1204, both of which had 24 mM ammonium sulfate and 8 mM
potassium nitrate but different base salts, (MS basal salts no nitrogen
and B5 major salts modified, respectively; Table 1)was softer than callus
produced on medium with 1mM ammonium sulfate and 24 mM potassium nitrate
(VM1445). Callus produced on medium VM1233 (Madhavi et al., Plant
Science, 131:95-103, 1998) was very compact and non proliferative.
Madhavi et al. showed that callus was subcultured on this medium for
three subculture periods each at three week intervals, although the
quality of the callus on this medium was not discussed in that reference.
Subculturing callus using the conditions of Madhavi et al. produced the
same initial results as those described in the reference, but it was
noted that with every subculture the callus became hard and non
proliferative. Thus, the quality of the callus using medium VM1233
decreased with every subculture. The removal of the polyvinylpyrrolidone
(PVP; medium VM1204) from the VM1233 medium helped to make the callus
soft. On media VM1491 and DC1152, 50% and 10% of the explants produced
sustainable callus, respectively. Explants and calli were transferred to
fresh medium every 3 weeks. Once the calli were separated from explants,
calli that were very proliferative were subcultured every 2 weeks. Fast
growing cell lines were chosen for subculture. Continuous subculture
helped change the morphology of the callus to a more desirable friable
morphology.

[0029] Subculturing continuously on medium with higher ammonium sulfate
and lower potassium nitrate resulted in the callus becoming very brown
from the stress and it eventually stopped growing. This was evident from
the fact that medium VM1445, which had full strength Gamborg's B5 (B5)
medium, but did not have ammonium sulfate or potassium nitrate did not
show browning and eventual death. Media VM1196 and VM1204 were
discontinued after 9 weeks because of undesired browning of the callus.
Various media (Table 1) were tried in order to characterize a medium that
would support growth of callus sustainably, without browning and eventual
death. Medium VM1516 which had full strength MS salts showed very
proliferative and sustainable growth. When VM1445 and VM1516 were
compared, VM1516 gave the most proliferative calli and also helped change
the morphology from compact to granular and eventually friable callus.
Medium VM1516 also proved the best for sustainably maintaining callus
derived from V. myrtillus seedlings. VM1516 was also confirmed to be the
best medium for initiating new callus from various V. myrtillus seedling
explants, with a success rate of 83%.

Example 3

Callus Induction from Vaccinium myrtillus Tissue Collected from NCGR

[0030] This example describes methods and media formulations for
initiating and maintaining callus from various explants (derived from
berries, nodes, internodes, or leaves) derived from field-grown V.
myrtillus plants.

[0031] Mature leaves and stems, and immature berries were surface
sterilized, as discussed above. The plant parts were cut into small 5 mm
sections before explanting into media VM1516 and VM1491. Berries were cut
open under sterile conditions and the skin was placed on culture plates
with media. Any berry flesh was removed before explanting.

[0032] Culture plates were kept in darkness at 25° C. In general,
callus was observed 4 weeks after initially explanting materials on
VM1516 and 6 weeks on VM1491. With regard to leaf explants, there was a
53% callus induction rate overall. Callus from leaf explants was produced
in VM1491 (73% of initial explants) and in medium VM1516 (76% of initial
explants) and no callus was observed in medium VM1672 and TC1596. It was
observed that the callus produced in VM1516 was more vigorous than that
produced in VM1491. With regard to nodes, 47% of those explants produced
callus in VM1516. Internodes were placed on 3 different media VM1516,
VM1491 and TC1596. On VM1516, 51% explants produced callus, while only
20% produced callus on VM1491 and none on TC1596. Among the explants from
berries, 59% produced callus on VM1516.

[0033] Callus derived from V. myrtillus tissue was subcultured every 3
weeks on VM1516. This callus was very proliferative and friable cell
lines were selected for further maintenance. Calli derived from these
tissues were maintained on medium VM1516 for over eight months and have
demonstrated consistent proliferation without change in quality of the
callus.

[0034] Friable cell lines created as in example 2 were chosen for
initiation of suspensions. Cell suspensions were created by introducing 1
g (approx) of fresh 2 week old V. myrtillus seedling callus (prepared as
in Example 2) into 15 ml of liquid medium (VM1799, VM1831 or DC1151;
Table 2) in a sterile 125 ml Erlenmeyer flask. The flasks were covered
with sterile silicon (foam) caps and agitated at 120 revolutions per
minute (rpm) in a gyrotatory shaker. The suspensions were kept in
darkness at 23° C. To establish the cell culture, the spent medium
was removed and fresh medium was added every week for 2 subcultures. The
growth of cells was measured by the rate of carbohydrate consumed by
measuring the delta of refractive index (RI) (as measured by degrees of
BRIX (i.e., % BRIX)) of the medium. If the RI was less than or equal to
half of the initial RI of the medium, fresh medium was added to the
cells. If the RI was greater than half, fresh medium was only added after
2 weeks. The subcultures were transferred weekly or biweekly as deemed
necessary.

[0035] Cultures that formed as either granular or fine suspension of cells
were retained, while cultures that did not form suspension cultures were
discarded. Once the suspension culture was established (3-4 subculture
periods), 25-35% of the cells were transferred to flask with fresh medium
every week. Packed cell volume (PCV) and RI was recorded at every
subculture to measure cell growth.

[0037] Friable cell lines were chosen for initiation of suspensions. Cell
suspensions were created by introducing V. myrtillus callus (prepared as
in Example 3 from nodes, internodes, leaves, and berries) into liquid
medium (VM1933; Table 2) in sterile Erlenmeyer flasks. The flasks were
covered with sterile silicon (foam) caps and agitated at 120 revolutions
per minute (rpm) in a gyrotatory shaker. The suspensions were kept in
darkness at 25° C. To establish the cell culture, the spent medium
was removed and fresh VM1933 medium was added. The growth of cells was
measured by the rate of carbohydrate consumed by measuring the delta of
refractive index (RI) of the medium. If the RI was less than or equal to
half of the initial RI of the medium, fresh medium was added to the
cells. If the RI was greater than half, fresh medium was only added after
2 weeks.

Example 6

Optimization of Cell Growth

[0038] This example describes methods used to increase cell growth of
suspensions. Cell culture productivity increases as a function of the
rate of cell growth and the density at which cell growth stops. To
determine the optimal inoculation density, suspension cultures of
Vaccinium myrtillus cells were initiated with an inoculum size yielding a
starting cell density of 15% packed cell volume ("PCV") and 25% PCV and
allowed to grow for 7 days. Cultures initiated at a cell density of 15%
PCV did not reach maximal density within 7 days. Cultures initiated at a
cell density of 25% PCV in Medium VM1831 (Table 1.) doubled in density
(i.e., total cell volume) within 7 days and reached a maximal average
cell density of 45-50% PCV within 7 days with some cell line cultures
showed over 55-60% PCV at day 7. Cell selection helped to capture
cultures that reached a 45% PCV or more PCV within 7 days or less (a
rapidly growing cell culture). Cultures that took more than 7 days to
reach 45% PCV were discarded.

[0039] After careful selection of cells that showed high sugar consumption
and good growth (FIG. 1.) through a number of generations, it was seen
that the final cell density on day 7 was very high (PCV around
65.8±0.63) when flasks were initiated with a 25% inoculum. This
inhibited proper shaking of the cells in the 125 ml flasks with a working
volume of 40 ml. Therefore, optimization of inoculums size was required
again once the cell line improved through cell selection process.
Calculation of the doubling time indicated that a 20% PCV inoculums would
yield around 60% final PCV on day 7. Experimental data supported this and
showed there was a significant difference (P=0.008) in the final PCV
(59.9±0.77) but no deleterious effect (P=0.39) on production or
productivity. Hence the inoculums size was changed to 20% PCV.

Example 7

Optimization of Polyphenol Production from Bilberry Suspensions by Cell
Selection and Medium Optimization of Suspension Cell Culture

[0040] After optimization of growth, production of polyphenol production
was achieved by changing media formulation and additional criteria for
cell selection.

[0041] The carbohydrate consumption was rapid in the cultures with the
cultures reaching RI of 0 to 0.6 by day 7. Polyphenol and/or procyanidin
production in VM1831 was low possibly due to sugar starvation. The medium
VM1831 had 20 g/L of sucrose. Liquid media was optimized by adjusting
carbohydrate level to maintain cultures without nutrient starvation. A
new medium VM1933 (Table 1.) was formulated with 30 g/L of sucrose to
avoid sugar starvation of the cells. In this medium the RI went down to
between 0.8 and 1.0. The production values of polyphenols went up from
about 2-4 g/L of PCV at 20g/L sucrose to about 5-10 g/L of PCV at 60 g/L
sucrose within 4 subcultures and could be maintained at a high production
level. The production values of procyanidins went up from about 1-2 g/L
of PCV at 20 g/L sucrose to about 3-7 g/L of PCV at 30 g/L sucrose within
4 subcultures and could be maintained at a high production level.

[0042] The cell selection process where we selected for flasks that
produced higher than average polyphenols quantified by a high through at
each subculture allowed for further improvement in polyphenol and
procyanidin production levels.

Example 8

Detection and Confirmation of Polyphenol and Procyanidin Production in
Suspensions from Various Parts of Bilberry Seedlings

[0043] In this example we demonstrate that we have been able to produce
procyanidin from suspensions prepared as in examples 4 and 5 from all
parts (roots, hypocotyls, berries, cotyledons, stem and leaves) of the
plants. FIG. 3 shows the chromatogram showing various sources. We have
also been able to confirm that what we are seeing is procyanidins by
overlaying with confirmed cocoa procyanidin chromatograms (FIG. 4) that
show same retention times for each oligomer as in cocoa, which also show
additional isomers of dimer, trimer and tetramer in Bilberry. Also
running a UV absorption at 280 nm showed that the pattern was similar to
cocoa and also confirmed presence of procyanidin (FIG. 5).

Example 9

Extraction of Polyphenols from Vaccinium Callus Culture and Suspension
Cultures

[0044] This example describes methods developed for extracting polyphenols
from callus and suspension cells of Vaccinium cultures developed in
examples 1-5. Polyphenols were extracted from approximately 0.4 ml of
fresh cells from suspensions with 0.4 ml 70% (v/v) acetone with 0.5%
acetic acid. A robust high throughput method was used as follows: From
each flask of cell culture to be analyzed, the packed cell volume (PCV)
of the sample was recorded prior to transferring 0.4 ml into a 96- deep
well plate. The supernatant from each well was removed and discarded with
a plastic transfer pipette. Next, 0.4 ml of extraction solvent (70%
acetone, 29.5% water, 0.5% acetic acid) and a tungsten carbide bead were
added to each well, and the plate was placed on a Mixer Mill to grind the
cells at 18 Hz for 4 minutes. The plate was then placed in a centrifuge
and centrifuged at 6000 rpm for 4 minutes to separate the cells from the
extract.

Example 10

Preliminary Analysis of Polyphenol Production in Culture

[0045] The method used to carry out the procyanidin analysis reaction was
designed to approximate fairly closely the original Swain and Hillis (J.
SCI. Food Agric. 10:63, 1959) method and Porter et al. (Phytochemistry,
25(1):223, 1986) method. The butanol-HCl extraction assay was used to
measure polyphenols in the extracts of Vaccinium myrtillus suspended
cells. The polyphenols are hydrolyzed to the monomers of (-)-epicatechin
and cyanidin by combining 0.1 ml of aqueous acetone extract and 1.0 ml of
butanol-HCl reagent (95:5 v/v) and heating the solution at 75° C.
for 60 minutes in a Qiagen deep well block (Valencia, Calif., USA).
Presence of cyanidin in the hydrolyzed sample was observed by the
formation of a pink color. The absorbance at 520 nm was determined, and
procyanidin content was calculated based on the amount of cyanidin formed
using a calibration curve created using different concentrations of
procyanidin B2 purchased from Chromadex, Inc. (Irvine, Calif.). Brighter
pink color indicated higher concentration of procyanidins in suspension
cultures. Based on this method the procyanidin content of several
suspension cultures ranged from 1 g/L to 10 g/L.

[0046] Total polyphenol content of bilberry cell extracts was measured
using the Folin-Ciocalteau assay (Slinkard, K.; Singleton, V. L. Total
Phenol Analysis:Automation and Comparison with Manual Methods. American
Journal of Enology and Viticulture 1977, 28: 49-55). Cell culture
extracts, in 70% acetone with 0.5% acetic acid, were analyzed for total
polyphenol content by taking 25 μl extract and adding it to 0.975 ml
of water to dilute the sample prior to beginning the assay. For the
quantification of polyphenols, 20 μl of the diluted extract is added
to 0.790 ml water plus 50 μl of Folin-Ciocalteau reagent. The reaction
is then stopped by the addition of 150 μl sodium carbonate solution.
The resulting solution is measured at 765 nm and compared to a
calibration curve of various dilutions of gallic acid solution measured
by the same assay to determine the concentration of total polyphenols in
the cell extracts.

[0047] Bilberry cells (0.5 mL) without media or 50 mg of ground bilberry
cells were sampled in 2.0 ml of micro-tubes or 1.2 ml tubes in a 96 well
block from Qiagen, Inc. Appropriate volumes of acidic (0-2% of citric,
acetic or ascorbic) aqueous extraction solvent (30-80% of acetone,
ethanol, methanol) was added to each of the bilberry cell samples and
then placed into ultrasonicator or BeadMill to extract polyphenols and/or
procyanidins. The samples were centrifuged for 4 minutes at 6000 rpm (RCF
5996). The supernatant may be filtered with 0.45 um membrane filter and
diluted to 10× (if necessary) by using the same aqueous extraction
solvents prior to analysis. The leftover extracts were stored in -20
degree of freezer for further analyses.

[0050] The purpose of the analytical method is to detect the presence of
the ten different individual procyanidins in fresh bilberry cells or
freeze-dried cells. Detectable procyanidins are monomer, dimmers,
trimers, tetramers, pentamers, hexamers, heptamers, octamers, nonamers
and decamers.

[0052] A common problem in the use of plant cell cultures is obtaining
consistent production of target products (Kim et al., Biotechnol Prog.
20(6) 1666, 2004). Therefore, a key for successful large-scale plant cell
culture is to maintain stable productivity. A process to scale-up
suspensions of bilberry cell cultures from 125 ml flasks to 250 mls and
then 500 ml flasks was successfully conducted. The speed of the shakers
was optimized for 500 ml flasks to give the same kind of growth and
production numbers as in the 125 ml flasks. Three different shaker speeds
were tested--100, 110 and 120 RPM. The average PCV was 50˜55% at
seven days, which was about 2.5 times greater than the initial PCV level
of 20% for all the treatments. However, the production yield (PY) was
significantly high at 110 RPM when compared to 100 RPM with a P value of
0.005. Although the difference in PY was not significant between 110 RPM
and 120 RPM, the color in the 120 RPM flasks was slightly darker, leading
to choose 110 RPM as preferred shaker speed for 500 ml flasks. Every
seven days of culture, biomass, sugar concentration in medium, and
polyphenol and/or procyanidin productivity, were measured.

[0053] Feasibility of scale up to 2.8 L flasks is carried out, where
shaking speed (rpm) and shaker stroke size is optimized. This
successfully yields similar growth and production as in 125 ml and 500 ml
flasks.

[0054] The present disclosure may be embodied in other specific forms
without departing from its spirit or essential characteristics. The
described embodiments are to be considered in all respects only as
illustrative and not restrictive. The scope of the disclosure is,
therefore, indicated by the appended claims rather than by the foregoing
description. All changes which come within the meaning and range of
equivalency of the claims are to be embraced within their scope.